Francisella tularensis is a highly infectious bacterium responsible for tularemia, a disease whose pneumonic form has potentially lethal consequences in humans. Francisella virulence depends on its ability to survive and replicate inside macrophages of the infected host, where the bacterium down modulates the macrophage immune functions. The current model of Francisella intracellular fate is initial enclosure within a phagosome, followed by escape from this phagosome and then replication in the cytoplasm, but the bacterial determinants controlling these individual stages are unknown. We have been using cell biology-, bacterial genetics- and genomics-based approaches to further characterize Francisella intracellular trafficking, identify genes expressed at various stages of the intracellular cycle and assess their role in Francisella virulence. Using models of primary macrophage infection with F. tularensis, we have previously established the intracellular cycle of this pathogen, which involves rapid phagosomal escape (Checroun et al., 2006, PNAS, 103:14578;Chong et al., 2008, Infect. Immun., 76:5488) followed by extensive proliferation in the cytosol and autophagy-mediated reentry into the endocytic compartment at late stages of the cycle (Checroun et al., 2006, PNAS, 103:14578;Wehrly et al., 2009, Cell. Microbiol, 11:1128). In our efforts to understand how host factors modulate the Francisella intracellular cycle, we have shown that IFN&#947;activation of macrophages does not affect the ability of Francisella to disrupt its early phagosome, yet restricts cytosolic growth of the bacterium (Edwards et al., 2010, Microbiology, 156:327). Additionally, we have established that targeting of Francisella to opsonic receptors via either complement or IgG opsonization, a process relevant to nave or immune hosts, negatively affects phagosomal escape and cytosolic proliferation, demonstrating that non-opsonized phagocytic pathways are more permissive to Francisella survival and proliferation than opsonic uptake processes (Geier and Celli, 2011, Infect. Immun., 79:2204). In our studies of the molecular mechanisms of Francisella intracellular pathogenesis, we have examined the functions encoded within the Francisella Pathogenicity Island (FPI), a locus required for virulence, and demonstrated in collaboration with Dr Karl Klose (University of Texas at San Antonio) that it encodes a functional Type 6 secretion system required for phagosomal escape (Barker et al., 2009, Mol. Microbiol. 74:1459). We have identified and characterized Francisella genes that are novel determinants of intracellular pathogenesis, through intracellular transcriptional profiling of the prototypical virulent strain Schu S4 of F. tularensis (Wehrly et al., 2009, Cell. Microbiol, 11:1128), among which two proteins encoded by these loci, DipA (FTT0369c) and FlpA (FTT1676), are surface-exposed during the intracellular cycle and required for cytosolic replication. The characterization of mutants in either dipA or flpA has revealed that cytosolic replication-deficient Francisella are captured by autophagy and cleared, indicating that this bacterium must interfere with autophagic recognition during its cytosolic phase. Additionally, mutants in either dipA or flpA conferred high levels of protection of mice against a pulmonary challenge with a virulent strain, suggesting that such mutants have some potential as genetically defined, live vaccine strains against tularemia. A patent application for these stable, genetically defined attenuated strains (No. PCT/US10/25417) has been submitted.